À la Fizeau in flow: Pulse shape‐assisted fluorescence lifetime

Abstract

Key term fluorescence resonance energy transfer FLUORESCENCE lifetime measurements are utilized more and more lately in molecular and cell biology partly due to its applicability for monitoring molecular dynamics and discrimination of molecular species, partly due to the commercial availability of microscopes and fluorimeters capable for fluorescence lifetime detection (1,2). Fluorescence lifetime is the average time an excited fluorophore spends in the excited state prior to returning to the ground state, via emission of light or other, non-radiative ways of de-excitation. Its widespread utility rests on the property that in addition to fluorescence, many other processes involving the excited fluorophore may exist which take place on the same time scale and may compete with fluorescence in the de-excitation and consequently may influence fluorescence lifetime (3-5). To name a few, these processes are: internal conversion, vibrational relaxation, intersystem crossing, F€ orster resonance energy transfer (FRET), dynamic quenching, solvent relaxation, charge transfer, and photolysis (photobleaching); and most of these processes involve interactions of the excited fluorophore with its local environment (1,3). For this reason fluorescence lifetime can be used to monitor locally the concentrations of ions and molecules, electric field and polarity, temperature, viscosity, and refractive index (1-3,6-9). By spanning a much larger range than the wavelength of the emitted fluorescence, it is more amenable for multiplexing than wavelength (8,10). It can be used as a contrast parameter, for example, for discriminating between emitters having overlapping emission spectra (9). It is also a state parameter, meaning that it is independent of conditions of excitation such as wavelength, intensity, and polarization of the exciting light. Based on its independence of concentration and due to the fact that it does not necessitate calibration, it is a more sensitive indicator of FRET than the intensity alone (3,11). It can be changed only physically-termed radiative decay engineering-by influencing the mode structure of the random vacuum field fluctuations responsible for the stimulation of spontaneous emission, for example, by placing a metal mirror in the vicinity of the fluorophore or by embedding the fluorophore in a cavity resonator (12,13).The detection of fluorescence lifetime rests on the "drag" and attenuation imposed on the reemission process, the fluorophore acting as a low pass filter, by cutting and smoothing out sharp features of the original time variation of the exciting light ( Fig. 1) (3). This feature mathematically is expressed by the operation on the time profile of excitation and pulseresponse of fluorophore-called convolution-leading to a shift to the right on the time-axis and demodulation for the time profile of fluorescence.Instead of using short excitation pulses the fluorescence lifetime can also be measured by a long lasting observation of fluorescence, which is periodically modulated at a frequency ($...